Improving the production of carbamoyltobramycin by an industrial Streptoalloteichus tenebrarius through metabolic engineering

Abstract Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes. Key points • The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified. • An oxygenase TobO was identified within the tobramycin biosynthesis cluster. • TobO and TobR have significant effects on the synthesis of carbamoyltobramycin. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-024-13141-2.


Supplementary Figure
Figure S1.Construction of strains and plasmids for the elimination of byproduct apramycin.a Construction of inactivation plasmid pSpc260-△aprJ and the genotype of mutants Tb-△aprJ, Tb-△aprK, Tb-△aprQ, Tb-△aprI and Tb-△aprM.b Identification of Tb-△aprJ, Tb-△aprK, Tb-△aprQ, Tb-△aprI and Tb-△aprM by PCR.The template of lane 1 was from Tb genomes with a 2179 bp band, the template of lane 2 was from △aprK mutant genomes with a 1714 bp band, primers di-aprK-F and di-aprK-R were used in this part, consistent with the expected deletion of 465 bp fragment within the region of the aprK.The template of lane 3 was from Tb genomes with a 1942 bp band, the template of lane 4 was from △aprI mutant genomes with a 1153 bp band, primers di-aprI-F and di-aprI-R were used in this part, consistent with the expected deletion of 789 bp fragment within the region of the aprI.The template of lane 5 was from Tb genomes with a 1547 bp band, the template of lane 6 was from △aprJ mutant genomes with a 1002 bp band, primers di-aprJ-F and di-aprJ-R were used in this part, consistent with the expected deletion of 545 bp fragment within the region of the aprJ.The template of lane 7 was from Tb genomes with a 1740 bp band, the template of lane 8 was from △aprQ mutant genomes with a 910 bp band, primers di-aprQ-F and di-aprQ-R were used in this part, consistent with the expected deletion of 821 bp fragment within the region of the aprQ.The template of lane 9 was from Tb genomes with a 1902 bp band, the template of lane 10 was from △aprM mutant genomes with a 1010 bp band, primers di-aprM-F and di-aprM-R were used in this part, consistent with the expected deletion of 892 bp fragment within the region of the aprM.The template of lane 1 was from Tb-△aprJ genomes with a 1639 bp band, the template of lane 2 was from Tb-△aprJ-△tobO mutant genomes with a 939 bp band, primers di-tobO-F and di-tobO-R were used in this part, consistent with the expected deletion of 700 bp fragment within the region of the tobO.Table S1 Plasmids

Figure S2 .
Figure S2.Construction of strains and plasmids for TobR research.a Construction of inactivation plasmid pSpc260-△tobR.b Construction of overexpression plasmid pSpc8660-tobR.c Identification of △tobR mutant by PCR.The template of lane 1 was from Tb-△aprJ-△tobR mutant genomes with a 1035 bp band and template of lane 2 was from Tb-△aprJ genomes with a 1463 bp band, consistent with the expected deletion of 428 bp fragment within the region of the tobR.

Figure S4 .
Figure S4.Functional analysis of AprM through NCBI database

Figure
Figure S7.plDDT score for the TobR structure predicted by Alphafold 2.

Figure S8 .
Figure S8.The regulatory targets of TobR on tobramycin biosynthesis gene cluster analyzed by EMSA.The protein concentrations in the experimental groups were 5 μM.

Table S3： Analysis of the gene products of the tobramycin gene cluster of Streptoalloteichus tenebrarius Tb.
(Altschul et al. 1990e results of BlastP analysis(Altschul et al. 1990).